Epigenetic Enhancer Marks and Transcription Factor Binding Influence Vk Gene Rearrangement in Pre-B Cells and Pro-B Cells [VDJ-seq]

There has been no study to date that has examined genomic DNA (gDNA) and RNA Igkappa repertoires directly from the same set of cells. In this study we performed deep sequencing on Igkappa repertoire library preparations from both gDNA and RNA from the same sorted cells. This was done for both pre-B cells, where most Igkappa rearrangement takes places as well as pro-B cells where some B cells undergo early Igkappa rearrangement. In addition, we perfored gDNA repertoire analysis on iEkappa-/- pre-B cells. We find that some Vk genes display skewed DNA or RNA representiation relative to the other. We also find that pro-B cell Igkappa rearrangement is skewed to Jkappa-distal Vkappa genes. Lastly, we find that iEkappa-/- pre-B cells are not capable of rearranging to the most proximal Vkappa family genes. gDNA (DNeasy, Qiagen) or RNA (RNeasy, Qiagen) was prepared from C57BL/6 pre-B cells, C57BL/6 pro-B cells, and iEkappa-/- pre-B cells. gDNA was sonicated to 500 to 1000 bp fragments, end repaired, A-tailed and adapter ligated. Jkappa gene biontinylated primer extended products were pulled out with streptavidin. Sequential PCRs added index, barcode and flow cell binding sequences. The last PCR utilized NEBNext Dual indexing primers. RNA was converted to cDNA with Transcriptor high-fidelity reverse transcriptase (Roche). RNase treatments removed unwanted RNA. cDNA was then adapter ligated using bridge adapters. Sequential PCRs was performed as in gDNA. Libraries were sequenced using Illumina MiSeq 2x300 read length.